Reperfusion duration distinctively alters apoptosis, necroptosis, ferroptosis, pyroptosis, and autophagy markers during early reperfusion in isolated rat hearts

Reperfusion injury is a paradoxical phenomenon involving mitochondrial dysfunction and cell death that reduces cardiac performance once blood flow is restored after myocardial infarction (MI). While several cell death mechanisms have been implicated, their specific contributions at different stages of reperfusion in the ex vivo ischemia–reperfusion (IRI) model remain unclear. This study assessed apoptosis, necroptosis, ferroptosis, autophagy, and pyroptosis at multiple reperfusion time points in rat hearts. Isolated hearts from adult male Sprague Dawley rats were perfused via the Langendorff retrograde method using Krebs–Henseleit solution, subjected to 30 min of equilibration, 25 min of global ischemia, and then 5, 30, or 60 min of reperfusion. Cardiac functional parameters were measured during perfusion, and mitochondria were isolated and analyzed for respiration, Ca 2+ -induced swelling, and ROS production. Cell death pathway markers were quantified via SDS-PAGE and western blotting. Cardiac physiological parameters, measured by LVDP and RPP, decreased significantly during reperfusion compared to pre-ischemic values. Mitochondrial State 3 oxygen consumption declined significantly by 38.7% as reperfusion progressed, while ROS production and Ca 2+ -induced swelling significantly increased 2.30- and 2.44-fold, respectively. Apoptosis showed a time-dependent 1.35-fold increase, as evidenced by Caspase-3 cleavage. Glutathione peroxidase 4, an endogenous ferroptosis inhibitor, decreased significantly by 43% during early reperfusion. Autophagy markers p62 and LC3B showed significant time-dependent increases of 2.08- and 1.37-fold, respectively. Necroptosis and pyroptosis markers showed no significant change during 60 min of reperfusion. Our results show an association between cardiac dysfunction and mitochondrial alterations, as well as differential activation of RCD markers during the early reperfusion phase. This suggests that specific mechanisms may predominate over others at different stages of reperfusion, indicating a time-sensitive interaction between these pathways that could influence future therapeutic strategies for MI patients.